Fgf receptor ligands for treating diabetes and obesity

ABSTRACT

Methods for treating diabetes or obesity are provided comprising administering to the subject an amount of an agent that binds a central nervous system FGF receptor homodimer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/181,413, filed Jun. 18, 2015, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

All publications, patents, patent application publications and booksreferred to herein, including those referred to by number in bracketsand recited at the end of the specification, are each herebyincorporated by reference in their entirety into the subject applicationto more fully describe the art to which the subject invention pertains.

Obesity and diabetes are disorders which have reached epidemicproportions worldwide. It is estimated that at least 25% of the adult USpopulation is overweight, while type 2 diabetes affects about 9.3% ofthe US population. Effective therapies for weight control are limited inefficacy. Diabetes medications meet with limited patient compliance.Thus, there is a very large demand for effective treatments readilyaccepted by the patient population.

Herein is disclosed a method for treating diabetes or obesity using anagent that binds a central nervous system FGF receptor homodimer.

SUMMARY OF THE INVENTION

A method of treating diabetes or obesity in a subject comprisingadministering to the subject an amount of an agent that binds a centralnervous system FGF receptor homodimer effective to treat diabetes orobesity in a subject.

A method of improving glucose tolerance in a subject comprisingadministering to the subject an amount of an agent that binds a centralnervous system FGF receptor homodimer effective to improve glucosetolerance in a subject.

A method of treating a prediabetes hyperglycemic episode in a subjectcomprising administering to the subject an amount of an agent that bindsa central nervous system FGF receptor homodimer effective to treat aprediabetes hyperglycemic episode in a subject.

Also provided is a method of treating diabetes or obesity in a subjectcomprising administering to the subject an amount of an agent comprisinga peptide having the sequence X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10,wherein F is a phenylalanine, V is a valine, Q is glutamine, X1 is theamino acid P or V, X2 is the amino acid N or D, X3 is the amino acid T,N or R, X4 is the amino acid Q or I, X5 is the amino acid H or Y, X6 isthe amino acid R or E, X7 is the amino acid E, D or N, X8 is the aminoacid S, G or T, X9 is the amino acid L, A or R, and X10 is the aminoacid V, M or A (SEQ ID NO:1) effective to treat diabetes or obesity in asubject.

Also provided is a method of treating a prediabetes hyperglycemicepisode in a subject comprising administering to the subject an amountof an agent comprising a peptide having the sequenceX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10, wherein F is a phenylalanine, V isa valine, Q is glutamine, X1 is the amino acid P or V, X2 is the aminoacid N or D, X3 is the amino acid T, N or R, X4 is the amino acid Q orI, X5 is the amino acid H or Y, X6 is the amino acid R or E, X7 is theamino acid E, D or N, X8 is the amino acid S, G or T, X9 is the aminoacid L, A or R, and X10 is the amino acid V, M or A (SEQ ID NO:1)effective to treat a prediabetes hyperglycemic episode in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A model for antagonism between FGF8 and FGF19/15 at FGFreceptors. FGF8 is represented by a diamond and binds to homodimeric FGFreceptors (dark-colored membrane spanning receptor) directly. FGF19/15is represented by an oval and binds to klotho (light-colored membranespanning receptor) directly which then forms a heteromeric complex withFGF receptors.

FIG. 2A-2E. ERK1/2 signaling involved in FGF19-glucose metabolismregulation. (2A) GTT with 2 g/kg of glucose (intraperitoneal injection)was performed after 6 hours of fast on HFD mouse males treated with icyinjection of vehicle or U0126 (2B) GTT (2 g/kg) was performed after 6hours of fast on HFD males treated icy with vehicle+FGF19 orU0126+FGF19. Mice were treated twice with FGF19 (starting 16 hours afterfasting and 6.5 hours apart) and tissues were collected 90 minutes afterthe last ip injection (2C) Analysis of co-localization between Npy-hrGFPpositive neurons (lighter gray) and p-ERK1/2 positive cells (darkergray) in 24 h fasted ob/ob Npy-hrGFP mice following intraperitonealinjections of FGF19. (2D) Quantification of Npy-hrGFP positive neuronsthat colocalize with pERK1/2 staining in ARC (Bregma −1.7 to −1.9 mm) ofob/ob Npy-hrGFP mice (10 sections from 3 mice were examined in eachgroup). (2E) Representative sections showing absence of co-localizationbetween Pomc-eGFP positive neurons (lighter gray) and p-ERK1/2 positivecells (darker gray) in 24 h fasted ob/ob Pomc-eGFP mice followingintraperitoneal injections of FGF19. *P<0.05, ***P<0.005.

FIG. 3. Reduced fat mass and improved glucose tolerance due to absenceof FGFR2 in hypothalamic AGRP/NPY neurons. Male mice (controls andAGRP:Fgfr2−/−) were placed on a high fat diet at 4 weeks of age (n=4-5per group) and analyzed at 16 weeks of age. Body compositions (toppanel) were determined by nuclear magnetic resonance spectroscopy withonly fat mass showing a significant difference. Glucose tolerance tests(middle panel) were performed after an overnight fast with a glucoseload of lmg/g body weight. Insulin tolerance tests (bottom panel) weredone in the post-prandial state. Data are presented as means andstandard deviations. An asterisk signifies a statistically significantdifference (p<0.05; t-test).

FIG. 4. Alignment of FGF8 family member isoforms highlighting thepresence of a conserved phenylalanine among FGF8 family b isoforms. Thealignment shows a phenylalanine (F) that differentiates between FGF8aand FGF8b biological activity. Structural analysis of FGF8b indicatedthat only the phenylalanine was in an ordered state when bound to FGFR1.The 13aa peptide used for probing glucose control in mice is underlinedfor each. (SEQ ID NO:7-11, top to bottom, respectively).

DETAILED DESCRIPTION OF THE INVENTION

A critical site of action for the metabolic effects of FGF19 isidentified herein to be the hypothalamus. Based on data derived fromdelivery of bile acids and FGF19 in combination with the impact ofablating expression of FGF receptors within the hypothalamus, aframework is provided wherein competition between FGF receptors inhomodimers and heterodimers (with Klotho co-receptors) can bemanipulated to improve metabolic status. Various FGF-derived peptideshave effects on body mass, body composition and glucose metabolism.Agents are disclosed, including peptides, that will lower body fat andimprove glucose tolerance.

A method of treating diabetes or obesity in a subject comprisingadministering to the subject an amount of an agent that binds a centralnervous system FGF receptor homodimer effective to treat diabetes orobesity in a subject.

A method of improving glucose tolerance in a subject comprisingadministering to the subject an amount of an agent that binds a centralnervous system FGF receptor homodimer effective to improve glucosetolerance in a subject.

A method of treating a prediabetes hyperglycemic episode in a subjectcomprising administering to the subject an amount of an agent that bindsa central nervous system FGF receptor homodimer effective to treat aprediabetes hyperglycemic episode in a subject.

In an embodiment of the methods, the agent does not bind an centralnervous system FGF receptor—Klotho heterodimer, and/or the agentprevents a Klotho binding to an FGF receptor to which the agent isbound.

In an embodiment of the methods, the agent comprises a peptidecomprising a phenylalanine residue.

Also provided is a method of treating diabetes or obesity in a subjectcomprising administering to the subject an amount of an agent comprisinga peptide having the sequence X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10,wherein F is a phenylalanine, V is a valine, Q is glutamine, X1 is theamino acid P or V, X2 is the amino acid N or D, X3 is the amino acid T,N or R, X4 is the amino acid Q or I, X5 is the amino acid H or Y, X6 isthe amino acid R or E, X7 is the amino acid E, D or N, X8 is the aminoacid S, G or T, X9 is the amino acid L, A or R, and X10 is the aminoacid V, M or A (SEQ ID NO:1) effective to treat diabetes or obesity in asubject. In an embodiment, the agent comprsing the peptide does notcomprise any further N-terminal amino acid(s) linked toX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10. In an embodiment, the agentcomprsing the peptide does not comprise any further C-terminal aminoacid(s) linked to X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10. In anembodiment, the agent comprsing the peptide does not comprise anyfurther N-terminal amino acid(s) or C-terminal amino acid(s) linked toX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10.

Also provided is a method of treating a prediabetes hyperglycemicepisode in a subject comprising administering to the subject an amountof an agent comprising a peptide having the sequenceX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10, wherein F is a phenylalanine, V isa valine, Q is glutamine, X1 is the amino acid P or V, X2 is the aminoacid N or D, X3 is the amino acid T, N or R, X4 is the amino acid Q orI, X5 is the amino acid H or Y, X6 is the amino acid R or E, X7 is theamino acid E, D or N, X8 is the amino acid S, G or T, X9 is the aminoacid L, A or R, and X10 is the amino acid V, M or A (SEQ ID NO:1)effective to treat a prediabetes hyperglycemic episode in a subject. Inan embodiment, the agent comprsing the peptide does not comprise anyfurther N-terminal amino acid(s) linked toX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10. In an embodiment, the agentcomprsing the peptide does not comprise any further C-terminal aminoacid(s) linked to X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10. In anembodiment, the agent comprsing the peptide does not comprise anyfurther N-terminal amino acid(s) or C-terminal amino acid(s) linked toX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10.

In an embodiment of the methods, the agent comprises a peptide havingthe sequence X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10, wherein F is aphenylalanine, V is a valine, Q is glutamine, X1 is the amino acid P orV, X2 is the amino acid N or D, X3 is the amino acid T, N or R, X4 isthe amino acid Q or 1, X5 is the amino acid H or Y, X6 is the amino acidR or E, X7 is the amino acid E, D or N, X8 is the amino acid S, G or T,X9 is the amino acid L, A or R, and X10 is the amino acid V, M or A (SEQID NO:1).

In an embodiment of the methods, X1 is P, X2 is N, X4 is Q, and X6 is R(SEQ ID NO:2).

In an embodiment of the methods, X3 is T or N, X7 is E or D, X8 is S orG, X9 is L or A, and X10 is V or M (SEQ ID NO:3).

In an embodiment of the methods, the agent comprises PNFTQHVREQSLV (SEQID NO:4). In an embodiment the agent does not comprise SPNFTQHVREQSLV.(SEQ ID NO:12)

In an embodiment of the methods, the agent comprises PNFNQYVRDQGAM (SEQID NO:5). In an embodiment the agent does not comprise SPNFNQYVRDQGAM(SEQ ID NO:13)

In an embodiment of the methods, the agent comprises VDFRIHVENQTRA (SEQID NO:6). In an embodiment the agent does not comprise NVDFRIHVENQTRA(SEQ ID NO:14).

In an embodiment of the methods, the agent comprises a peptideX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 having 90% or more sequenceidentity to (SEQ ID NO:4). In an embodiment of the methods, the agentcomprises a peptide X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 having 90% ormore sequence identity to (SEQ ID NO:5). In an embodiment of themethods, the agent comprises a peptideX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 having 90% or more sequenceidentity to (SEQ ID NO:6). In an embodiment of the methods, the agent is13 amino acids in length.

In an embodiment of the methods, the agent comprises a peptideX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 having 95% or more sequenceidentity to (SEQ ID NO:4). In an embodiment of the methods, the agentcomprises a peptide X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 having 95% ormore sequence identity to (SEQ ID NO:5). In an embodiment of themethods, the agent comprises a peptideX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 having 95% or more sequenceidentity to (SEQ ID NO:6). In an embodiment of the methods, the agent is13 amino acids in length.

In an embodiment of the methods, the agent comprises a peptide linked toa molecular entity which increases plasma half-life of the peptide. Inan embodiment, the molecular entity which increases plasma half-life ofthe peptide is not itself a peptide. In an embodiment of the methods,the molecular entity is a polyethylene glycol (PEG).

In an embodiment, the molecular entity which increases plasma half-lifeof the peptide is an albumin-binding peptide attached to the peptidehaving the sequence X1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 via a linker ordirectly. In an embodiment, the molecular entity which increases plasmahalf-life of the peptide is an albumin-binding antibody fragmentconjugated to the peptide having the sequenceX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10 via a linker or directly.

In an embodiment of the methods, the peptide is acylated. In anembodiment of the methods, the peptide is not acylated.

In an embodiment of the methods, the agent is administered in apH-buffered solution. In an embodiment of the methods, the agent isadministered in a sterile solution. In an embodiment of the methods, theagent is administered in a sterile pH-buffered solution. In anembodiment of the methods, the pH-buffered solution comprises anartificial pH-buffer.

In an embodiment of the methods, the agent is administered in a mannersuch that it can enter the central nervous system of the subject.

In an embodiment of the methods, the agent is administered systemicallyto the subject.

In an embodiment of the methods, the agent is administered parenterallyto the subject.

In an embodiment of the methods, the agent is administered to nasalepithelia of the subject.

In an embodiment of the methods, the agent is administered by injectioninto the subject. In an embodiment of the methods, the agent isadministered by subcutaneous injection into the subject.

In an embodiment of the methods, the agent is administered directly tothe central nervous system of the subject.

In an embodiment of the methods, the agent is administered weekly. In anembodiment of the methods, the agent is administered biweekly. In anembodiment of the methods, the agent is administered monthly. In anembodiment of the methods, the agent is administered daily.

In an embodiment of the methods, the agent is administered from animplanted device that delivers the agent from a reservoir. In anembodiment the reservoir is a dry reservoir. In an embodiment of themethods, the agent is administered from an implanted device thatdelivers the agent from a reservoir via an osmotic pump mechanism. In anembodiment of the methods, the agent is chronically administered from animplanted device that delivers the agent from a reservoir.

In an embodiment of the methods, the method is to treat diabetes. In anembodiment of the methods, the diabetes is type II diabetes (also calledtype 2 diabetes). In an embodiment of the methods, the method is totreat obesity. In an embodiment of the methods, the method treatsprediabetes. In an embodiment of the methods, the method treatsprediabetes hyperglycemia.

As used herein, “treating” a diabetes means that one or more symptoms ofthe disease, such as the diabetes itself, or a resultant symptom of thediabetes such as blindness, heart damage, lower limb ischemia or otherparameters by which the disease is characterized, are reduced,ameliorated, inhibited, placed in a state of remission, or maintained ina state of remission. In an embodiment, the method inhibits furtherdevelopment of the diabetes. In an embodiment, the method inhibitsfurther development of a pathology that results from the diabetes.

The invention also provides a method of treating a prediabetes transienthyperglycemic episode. As used herein, “treating” a prediabetestransient hyperglycemic episode means that one or more symptoms of thedisease, such as damage resulting from hyperglycemia (e.g. tissuedamage), or other parameters by which the disease is characterized, arereduced, ameliorated, inhibited, or maintained in a state ofprediabetes. In an embodiment, the method inhibits further developmentof the prediabetes into diabetes. In an embodiment, the method inhibitsfurther development of a pathology that results from the prediabeteshyperglycemia. In an embodiment, a prediabetes transient hyperglycemicepisode is considered a transient hyperglycemia of blood sugar >180mg/dL to >240 mg/dL. In an embodiment, a prediabetes transienthyperglycemic episode is considered a transient hyperglycemia of bloodsugar >200 mg/dL.

As used herein, “treating” obesity in a subject who has obesity means tostabilize, reduce, ameliorate or eliminate a sign or symptom of obesityin the subject. In an embodiment, obesity as used herein ischaracterized by the subject having a body mass index of 30.0 or greater(and thus includes the states of significant obesity, morbid obesity,super obesity, and super morbid obesity). In regard to gender, womenwith over 30% body fat are considered obese, and men with over 25% bodyfat are generally considered obese. The methods of treating obesity asdisclosed herein are also applicable to treating an overweight state ina subject, defined as a body mass index of the subject of from 25.0 to29.9, so as to stabilize, reduce, ameliorate or eliminate a sign orsymptom of the overweight state in the subject.

In an embodiment, the agent that binds a central nervous system FGFreceptor homodimer effective to treat diabetes or obesity in a subjectinhibits or antagonizes the FGF receptor homodimer.

Klotho is a type-I membrane protein (e.g. RefS eq. NP_004786) encoded inhumans by the KL gene (e.g. NM_004795). In an embodiment of the methods,the Klotho is a human Klotho.

In an embodiment of the methods, the FGF receptor is a human FGFreceptor (human FGFR). In an embodiment of the methods, the FGF receptoris a type IIIc isoform. In an embodiment of the methods, the FGFreceptor is a FGFR1. In an embodiment of the methods, the FGF receptoris a FGFR2.

There are 21 isoforms of FGFR1 (principally due to splicing) in humans.The UNIPARC identifiers for these 21 protein isoforms are(UniProtKB/Swiss-Prot) P11362-1 through P11362-21. Each of these is partof an individual embodiment of the invention where FGFR is recited.Similarly, there are 10 protein isoforms for FGFR2 with accession numberP21802 (UniProtKB/Swiss-Prot). Each of these is part of an individualembodiment of the invention where FGFR is recited.

Both FGFR1 and FGFR2 bind FGF8 with high affinity, FGFR1 more so. Thesplice isoforms, which are also encompassed by the invention, are theIIIc forms of both receptors. The brain is highly enriched in the IIIcisoforms.

This invention will be better understood from the examples follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims that follow thereafter.

EXAMPLE 1

Fibroblast growth factor (FGF) signaling has been implicated in theregulation of energy balance along with glucose and lipid homeostasis.Two of the three endocrine FGFs, FGF19 and FGF21, but not FGF23, havedemonstrated capacities to prevent or ameliorate diet induced obesity,improve glucose homeostasis and reduce hepatosteatosis (1,2). Whileperipheral organs have been suggested as important targets of FGFeffects, it has been demonstrated that FGF19 affects the central nervoussystem to reduce body weight and improve glucose control (3).Melanocortins are critical mediators of FGF19's effects. It was shownthat FGF19 activated ERK1/2 in hypothalamic AGRP/NPY neurons, but FOSwas reduced. Moreover, FGF19 prevented the induction of AGRP and NPYmRNA in mice fasted ovemight. Interestingly, no effects of FGF19 wereseen on POMC neurons. In its actions, FGF19 appears to be a partialleptin mimetic by activating signaling pathways in hypothalamic AGRP/NPYneurons that are downregulated in leptin resistant states.

Using genetic models of ablating FGF receptors within hypothalamicAGRP/NPY neurons it was predicted that ablation of FGFR1 and/or FGFR2 inhypothalamic AGRP/NPY neurons would result in obese and insulinresistant mice. However, studies of two genetic models, AGRP:Fgfr1−/−and AGRP:Fgfr2−/−, indicate that ablation of FGF receptor signalingprevents weight gain and improves glucose tolerance. These data indicatethat both FGFR1 and FGFR2 have metabolic actions. One explanation isthat these effects are due to compensatory effects as the geneticablations occur during a period of high plasticity within the CNS.Alternatively, these data support the possibility of a second ligand (analternative or alternative set of ligands) for hypothalamic FGFR1 andFGFR2, in addition to FGF19. These data could be explained by theability of FGF receptors to form homodimers in response to conventionalFGFs and FGFR-Klotho heterodimers to signal binding of endocrine FGFs.

FGFR1 and FGF8 constitute a receptor/ligand pair that exerts majoreffects on brain development (4). While total absence of FGFR1 and FGF8are lethal, partial deficiencies in humans are associated with defectivedevelopment of the olfactory bulb and gonadotrophin hormone releasinghormone (GnRH) neurons (5). Metabolic disturbances in patients withmutant FGFR1 or FGF8 have not been reported although major confoundingeffects of altered sex hormone production and altered pubertaldevelopment should be taken into account in interpreting the clinicalpresentations of such individuals. FGF8, along with FGF17 and FGF18(FGF8 subfamily), are secreted proteins which are localized by theirheparan sulfate binding regions to function as patterning and guidingsignals for neurons. FGF8 binds directly to a groove conserved betweenFGFR1, FGFR2 and FGFR3 (6). This groove also binds Klotho and betaKlotho, co-receptors for FGF receptors that avidly bind FGF19 and FGF21.Thus, there is competitive binding between FGF8 and Klotho to FGFRs,providing a structural basis for antagonism between FGF8 and FGF19.Moreover, FGF8 has multiple splice isoforms with the main forms beingFGF8a and FGF8b that apparently have antagonistic actions (7). Thismodel is relevant only to FGF receptors of the IIIc isoforms which areprincipally expressed in the brain. Removal of the heparan sulfatebinding domain may prolong retention time although biological activityis likely to be reduced (but not eliminated). Also, FGF8 is theprototype of a gene family that includes FGF17 and FGF18. Thus, thereare multiple means of manipulating FGF8 signaling due to the intrinsiccomplexity of its biology.

Central FGF19 effects on glucose metabolism rely on functional ERK1/2signaling: It was previously shown that FGF19, injected centrally andperipherally, could improve glucose handling in obese mice (ob/ob) andhigh fat fed mice (3). It was assessed whether increased ERK1/2activation could mediate the glucose lowering effect of FGF19. To thataim, the inhibitor U0126 was used on HFD-fed animals. Importantly, itwas first determined that a dose of 5 μg of U0126 infused in the 3rdventricle of HFD-fed males did not impact on glucose clearance during aGTT, to exclude any effect of U0126 itself on glucose metabolism (FIG.2). Then, glucose tolerance was compared between HFD-fed mice that werepre-treated with vehicle or with U0126 thirty minutes before icyinjection of FGF19. It was found that the pharmacological blockade ofhypothalamic phosphorylation of ERK1/2 with U0126, blocked the glucoselowering effect of FGF19.

The neuronal population targeted by FGF19 was investigated. 24 h fastedNpy-hrGFP transgenic mice deficient in leptin (Npy-hrGFP ob/ob), inwhich AGRP/NPY neurons in ARC appear GFP+, were injectedintraperitoneally with FGF19 and the colocalization betweenphospho-ERK1/2 and Npy-GFP was analyzed in ARC. Interestingly, it wasfound that FGF19-induced pERK1/2 staining colocalized with NPY-GFP+neurons. A similar experiment performed in Pomc-eGFP ob/ob mice (POMCneurons appear GFP positive) showed that Pomc-eGFP positive neurons donot colocalize with pERK1/2 staining in ARC. Overall, the data supportthat central FGF19 improved glucose metabolism required activation ofERK1/2 signaling and that AGRP/NPY neurons are a likely mediator ofFGF19 action.

Loss of FGFR2 in AGRP/NPY neurons improves metabolic status: Data frommice (AGRP:Fgfr2−/−; genotype Fgfr2-flox/flox Agrp-CRE) shows expressionof Fgfr2 is specifically ablated in hypothalamic AGRP neurons. Similardata were obtained from AGRP:Fgfrl mice (data not shown) although theeffects were observed on standard mouse chow and accentuated on high fatchow. After 3 months on a high fat diet, lower weight gain was observedin AGRP:Fgfr2 male mice compared to controls (Fgfr2-flox/flox). Glucosetolerance and insulin sensitivity were also improved in mice withoutFGFR2 in AGRP neurons. Interestingly, the Kd of FGFR2 for FGF8b (6) is ˜twice that of FGFR1, providing a mechanism for the reduced efficacy ofthe FGFR2 ablation to impart DIO-resistance, relative to FGFR1 ablation.These data indicate that FGFR2 signaling in AGRP/NPY neurons areimportant for appropriate glucose homeostasis although the a prioriprediction would have pointed to a worsened metabolic state in theabsence of FGFR2. These conflicting data are reconciled in the modelsuggested in the previously presented model of antagonism between FGFligands at FGF receptor homodimers and heterodimers.

Defining the effect of altering FGF receptor signaling in hypothalamicAGRP/NPY neurons after conditional expression of FGFR1 dominant negativeand constitutive active lentivirus: Lentiviral constructs were developedfor conditional expression of FGFR1 constructs that are dominantnegative (FGFR1 wherein the entire intracytoplasmic tyrosine kinasedomain is replaced by mCHERRY (8,9)) and constitutive active (fusion ofMyc and the FGFR1 kinase domain bearing a point mutation [Lys656G1u]that confers high intrinsic kinase activity (10) to activate SHP2,PLC-gamma, MAPK, PI3K and STATS). These lentiviral expression vectorsare delivered bilaterally to the mediobasal hypothalamus of AGRP-CREmice to activate expression of the FGFR1 constructs in AGRP/NPY neuronsdue to CRE mediated excision of a floxed transcriptional blockercassette upstream on the coding sequence for FGFR1. Mice are placed on ahigh fat diet for up to 12 weeks. Subsequently, body weight gain andglucose tolerance along with insulin sensitivity is determined.

Structures of FGFR1 lentivirus constructs:

-   Myr—myristoylaton signal from v-src; CMV—cytomegalovirus promoter;    mCherry—red shifted GFP. The virus has been tested by icy injection    into Agrp-IRES-CRE mice and mCherry fluorescence observed,    indicating successful CRE mediated expression of the FGFR1-mCherry    fusion gene.-   General construction of conditional FGFR1 within a lentivirus    vector:-   CMV promoter—loxP—polyA signal/transcriptional pause—loxP—FGFR1-   Dominant negative—dn-FGFR1 (SP—signal peptide; Ig—immunoglobulin    like domain; TM—transmembrane domain):-   CMV promoter—loxP—polyA/transcriptional    pause—loxP—SP-Ig1-Ig2-Ig3-TM-mCherry-   Constitutively active—ca-FGFR1 with K656E (activating) mutation:-   myr-Kinase1-Kinase2-mCherry

K656E

HEK 293 cells are transfected with the dnFGFR1-mCherry lentivirus. Cellsare fixed and visualized by fluorescence of the cytoplasmicallylocalized mCherry and nuclei stained with DAPI, a DNA-bindingfluorochrome. Each blue DAPI-stained nucleus is enveloped within a redmCherry-labeled cytoplasmic shell. Cells are positively selected withpuromycin as the lentivirus confers puromycin resistance.

Defining the effects of FGF8b and FGF19 and their derived peptides onenergy balance, body weight and glucose tolerance: Peptides derived fromFGF8b, FGF17b and FGF18 (underlined in FIG. 4) are used for theircapacity to improve glucose tolerance in high fat fed mice as well asob/ob B6 mice. A mutant of FGF8b wherein the phenylalanine was mutatedto an alanine (F61A) converted the peptide's activity spectrum to thatof FGF8a (7), indicating the crucial nature of F61 residue for FGF8bactivity. A mutated version of the FGF8b peptide with F61A is alsoemployed to determine if it is as ineffective to improve glucosehandling. Peptides are delivered by intraparenchymal injections into themedibasal hypothalamus. Insulin release and hepatic glucose productionare also assessed as the mechanism(s) for the improved glucoseparameters after FGF8b peptide administration. The ability of thepeptide when injected systemically to affect glucose handling is alsodetermined. High fat fed B6 mice are used for the systemic injections ofpeptide. Off target effects are unlikely due to the low affinity ofnon-111c FGF receptor isoforms (which are mainly expressed in theperipheral tissues) to bind FGF8b.

A peptide of 13 amino acids (PNFTQHVREQSLV) (SEQ ID NO:4) wassynthesized based on the conserved sequences between the FGF8 and FGF17b splice isoforms and the helical nature of this region of FGF8 based ona published crystal structure. This peptide was injectedintraparenchymally into the mediobasal hypothalamus of 8 month oldoutbred mice. Having been fasted overnight, the mice were subjected toan oral glucose tolerance test (2 mg/g of glucose) using a crossoverdesign to obtain a control condition. It was found that the FGF8bderived peptide improved glucose tolerance in mice when compared to thesame mice after injection with artificial cerebrospinal fluid (aCSF).With aCSF, the median area under the curve for the 3 hour GTT was 43,747mg-min/dL whereas it was 33,524 ng-min/dL with F8b13. Blood glucoseconcentrations were not different before the glucose load. These FGFderived peptide antagonists are useful as therapeutic agents in diabetesand obesity conditions.

REFERENCES

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1-4. (canceled)
 5. A method of treating diabetes or obesity in a subjectcomprising administering to the subject an agent comprising a peptidelinked to a molecular entity that increases the plasma-half-life of thepeptide, wherein the peptide consists of the sequenceX1-X2-F-X3-X4-X5-V-X6-X7-Q-X8-X9-X10, wherein F is a phenylalanine, V isa valine, Q is glutamine, X1 is the amino acid P or V, X2 is the aminoacid N or D, X3 is the amino acid T, N or R, X4 is the amino acid Q orI, X5 is the amino acid H or Y, X6 is the amino acid R or E, X7 is theamino acid E, D or N, X8 is the amino acid S, G or T, X9 is the aminoacid L, A or R, and X10 is the amino acid V, M or A (SEQ ID NO:1)effective to treat diabetes or obesity in a subject.
 6. (canceled) 7.The method of claim 5, wherein X1 is P, X2 is N, X4 is Q, and X6 is R(SEQ ID NO:2).
 8. The method of claim 7, wherein X3 is T or N, X7 is Eor D, X8 is S or G, X9 is L or A, and X10 is V or M (SEQ ID NO:3). 9-12.(canceled)
 13. The method of claim 5, wherein the molecular entity is apolyethylene glycol.
 14. The method of claim 5, wherein the agent isadministered in a manner such that it can enter the central nervoussystem of the subject.
 15. The method of claim 5, wherein the agent isadministered systemically to the subject.
 16. The method of claim 5,wherein the agent is administered parenterally to the subject.
 17. Themethod of claim 5, wherein the agent is administered to nasal epitheliaof the subject.
 18. The method of claim 5, wherein the agent isadministered by injection into the subject.
 19. The method of claim 5,wherein the agent is administered directly to the central nervous systemof the subject. 20-25. (canceled)
 26. The method of claim 5, wherein thediabetes is type II diabetes.
 27. (canceled)
 28. The method of claim 5,wherein the molecular entity is attached to the peptide via a linker.29. The method of claim 5, wherein the molecular entity is analbumin-binding peptide.
 30. The method of claim 5, wherein themolecular entity is an albumin binding antibody fragment.